| 1. |
- Chermenina, Maria, 1978-
(författare)
-
GDNF and alpha-synuclein in nigrostriatal degeneration
- 2014
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Parkinson’s disease is a common neurological disorder with a complex etiology. The disease is characterized by a progressive loss of dopaminergic cells in the substantia nigra, which leads to motor function and sometimes cognitive function disabilities. One of the pathological hallmarks in Parkinson’s disease is the cytoplasmic inclusions called Lewy bodies found in the dopamine neurons. The aggregated protein α-synuclein is a main component of Lewy bodies. In view of severe symptoms and the upcoming of problematic side effects that are developed by the current most commonly used treatment in Parkinson’s disease, new treatment strategies need to be elucidated. One such strategy is replacing the lost dopamine neurons with new dopamine-rich tissue. To improve survival of the implanted neurons, neurotrophic factors have been used. Glial cell line-derived neurotrophic factor (GDNF), which was discovered in 1993, improves survival of ventral mesencephalic dopamine neurons and enhances dopamine nerve fiber formation according to several studies. Thus, GDNF can be used to improve dopamine-rich graft outgrowth into the host brain as well as inducing sprouting from endogenous remaining nerve fibers. This study was performed on Gdnf gene-deleted mice to investigate the role of GDNF on the nigrostriatal dopamine system. The transplantation technique was used to create a nigrostriatal microcircuit from ventral mesencephalon (VM) and the lateral ganglionic eminence (LGE) from different Gdnf gene-deleted mice. The tissue was grafted into the lateral ventricle of wildtype mice. The results revealed that reduced concentrations of GDNF, as a consequence from the Gdnf gene deletion, had effects on survival of dopamine neurons and the dopamine innervation of the nigrostriatal microcircuit. All transplants had survived at 3 months independently of Gdnf genotype, however, the grafts derived from Gdnf gene-deleted tissue had died at 6 months. Transplants with partial Gdnf gene deletion survived up to 12 months after transplantation. Moreover, the dopaminergic innervation of striatal co-grafts was impaired in Gdnf gene-deleted tissue. These results highlight the role of GDNF for long-term maintenance of the nigrostriatal dopamine system. To further investigate the role of GDNF expression on survival and organization of the nigrostriatal dopamine system, VM and LGE as single or combined to double co-grafts created from mismatches in Gdnf genotypes were transplanted into the lateral ventricle of wildtype mice. Survival of the single grafts was monitored over one year using a 9.4T MR scanner. The size of single LGE transplants was significantly reduced by the lack of GDNF already at 2 weeks postgrafting while the size of single VM was maintained over time, independently of GDNF expression. The double grafts were evaluated at 2 months, and the results revealed that lack of GDNF in LGE reduced the dopamine cell survival, while no loss of dopamine neurons was found in VM single grafts. The dopaminergic innervation of LGE was affected by absence of GDNF, which also caused a disorganization of the striatal portion of the co-grafts. Small, cytoplasmic inclusions were frequently found in the dopamine neurons in grafts lacking GDNF expression. These inclusions were not possible to classify as Lewy bodies by immunohistochemistry and the presence of phospho-α-synuclein and ubiquitin; however, mitochondrial dysfunction could not be excluded. To further study the death of the dopamine neurons by the deprivation of GDNF, the attention was turned to how Lewy bodies are developed. With respect to the high levels of α-synuclein that was found in the striatum, this area was selected as a target to inject the small molecule – FN075, which stimulates α-synuclein aggregation, to further investigate the role of α-synuclein in the formation of cytoplasmic inclusions. The results revealed that cytoplasmic inclusions, similar to those found in the grafts, was present at 1 month after the injection, while impairment in sensorimotor function was exhibited, the number of dopamine neurons was not changed at 6 months after the injection. Injecting the templator to the substantia nigra, however, significantly reduced the number of TH-positive neurons at 3 months after injection. In conclusion, these studies elucidate the role of GDNF for maintenance and survival of the nigrostriatal dopamine system and mechanisms of dopamine cell death using small molecules that template the α-synuclein aggregation.
|
|
| 2. |
- Nikkhah, Guido, et al.
(författare)
-
Preservation of fetal ventral mesencephalic cells by cool storage : in-vitro viability and TH-positive neuron survival after microtransplantation to the striatum
- 1995
-
Ingår i: Brain Research. - : Elsevier BV. - 0006-8993. ; 687:1-2, s. 22-34
-
Tidskriftsartikel (refereegranskat)abstract
- Preservation of fetal ventral mesencephalic (VM) dopaminergic tissue prior to transplantation has been hampered by the fact that the cells are vulnerable to mechanical and osmotic stress after storage. Previous quantitative studies have shown that cool storage in a so-called 'hibernation medium' prior to grafting, can be used safely for up to 2 days without morphological or functional losses [16,32] using standard transplantation techniques. In the present study on rat fetal VM tissue we have investigated (i) the accuracy of different vital stains (trypan blue exclusion and ethidium bromide stain) to predict in vivo viability of VM cell suspensions after grafting; (ii) the influence of different storage media (glucose-saline, HBSS, DMEM, CO2-independent medium and hibernation medium), temperatures (+4 degrees C or +21 degrees C) and preparations (cell suspension or intact pieces) on the viability scores and total number of cells in vitro; and (iii) the survival and functional effects of intrastriatally grafted VM tissue after preservation by cool storage for up to 12 days using a less traumatic microtransplantation technique. The results show that cool storage at +4 degrees C of intact VM pieces in hibernation medium gives the best in vitro viability scores. Microtransplantation of cell suspensions prepared from cool-stored VM tissue produced good survival of tyrosine hydroxylase (TH)-positive graft neurons for up to 8 days of storage, and functional compensation in the amphetamine-rotation test for up to 12 days of storage. The total yield of surviving TH-positive neurons was unchanged, compared to fresh grafts, after 5 and 8 days of storage, and only reduced by 48% in the grafts stored for 12 days prior to implantation. These findings highlight the potential usefulness of a combination of cool storage and microtransplantation techniques to be able to extend the preservation periods of VM tissue. Such procedures may ultimately help to increase the safety and flexibility in experimental and clinical studies on neural transplantation of dopaminergic neurons.
|
|
| 3. |
- Ribeiro, Diogo, et al.
(författare)
-
Efficient expansion and dopaminergic differentiation of human fetal ventral midbrain neural stem cells by midbrain morphogens
- 2013
-
Ingår i: Neurobiology of Disease. - : Elsevier BV. - 0969-9961 .- 1095-953X. ; 49, s. 118-127
-
Tidskriftsartikel (refereegranskat)abstract
- Human fetal midbrain tissue grafting has provided proof-of-concept for dopamine cell replacement therapy (CRT) in Parkinson's disease (PD). However, limited tissue availability has hindered the development and widespread use of this experimental therapy. Here we present a method for generating large numbers of midbrain dopaminergic (DA) neurons based on expanding and differentiating neural stem/progenitor cells present in the human ventral midbrain (hVM) tissue. Our results show that hVM neurospheres (hVMN) with low cell numbers, unlike their rodent counterparts, expand the total number of cells 3-fold, whilst retaining their capacity to differentiate into midbrain DA neurons. Moreover, Wnt5a promoted DA differentiation of expanded cells resulting in improved morphological maturation, midbrain DA marker expression, DA release and electrophysiological properties. This method results in cell preparations that, after expansion and differentiation, can contain 6-fold more midbrain DA neurons than the starting VM preparation. Thus, our results provide evidence that by improving expansion and differentiation of progenitors present in the hVM it is possible to greatly enrich cell preparations for DA neurons. This method could substantially reduce the amount of human fetal midbrain tissue necessary for CRT in patients with PD, which could have major implications for the widespread adoption of this approach. (C) 2012 Elsevier Inc. All rights reserved.
|
|
